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CLASSIFICATION OF SMALL FLAT OYSTERS OF OSTREA STENTINA SPECIES COMPLEX AND A NEW SPECIES OSTREA NEOSTENTINA SP. NOV. (BIVALVIA: OSTREIDAE).

INTRODUCTION

Oysters are widely distributed in world oceans and estuaries. They are important fishery resources and important ecological service providers (Beck et al. 2011). Oysters support important aquaculture industries worldwide (Guo et al. 1999, Guo 2009). Despite their importance, the diversity and distribution of living oysters is not fully understood. Classification of oyster species has been problematic because of plasticity in shell morphology. Many oyster species have been misidentified, resulting in considerable confusion in classification (Harry 1985). Genetic studies over the past two decades have significantly improved oyster taxonomy, and about 100 species of living oysters are recognized (Bayne 2017, Guo et al. 2018). Still, many taxonomic uncertainties exist, and new species are to be discovered.

The ostreid genus Ostrea contains many fossils and living species. Harry (1985) revived the genus Ostreola (Monterosato, 1884), with the type species Ostreola stentina and member species Ostreola equestris and Ostreola conchaphila. Subsequently, Ostreola was reverted to Ostrea for lacking distinctive morphological characters (Harry 1985, Coan et al. 2000) and genetic support (Kirkendale et al. 2004, Lapegue et al. 2006, Shilts et al. 2007, Poison et al. 2009). Studies have indicated that some species of Ostrea/Ostreola are synonyms of O. stentina (Payraudeau, 1826). Ranson (1967) considered that subspecies O. stentina var. isseli (Bucquoy, Dautzenberg & Dollfus, 1887), O. stentina var. prepratxi (Bucquoy, Dautzenberg & Dollfus, 1887), and O. stentina syrica (Pallary, 1938), as well as Ostreola parenzani (Settepassi in Parenzan, 1974) and Ostreola curvata (Risso, 1826) were synonyms of O. stentina. Harry (1985) synonymized Ostrea spreta (d'Orbigny, 1846) from Southern Brazil and Caribbean Antillean islands with the northwest Atlantic/Caribbean Ostrea equestris (Say, 1834). Molecular study of O. equestris (Kirkendale et al. 2004) has revealed that the American O. equestris and New Zealand Ostrea aupouria (Dinamani & Beu 1981) are close sister taxa. Other studies (Shilts et al. 2007, Raith et al. 2015) included the Mediterranean/Eastern Atlantic--type species, O. stentina, and indicated that O. equestris and O. aupouria were synonyms of O. stentina. Salvi et al. (2014) also suggested that O. aupouria, O. equestris, O. spreta, and O. stentina may be one species. Hamaguchi et al. (2017) recently recorded O. stentina in Japan as part of the O. stentina complex that included O. stentina, O. aupouria, O. equestris, and O. spreta. It has been suggested that O. equestris is a separate species, which is different from two other species under the name O. stentina (Guo et al. 2018). Nevertheless, taxonomic status of the O. stentina complex is uncertain and requires further analysis, especially given its wide geographic distribution ranging from southern Mediterranean coasts, southwestern coast of the Iberian Peninsula, southern coast of Argentina, western and eastern Atlantic coasts, Gulf of California in eastern Pacific, and Asian Pacific. Taxonomic status of some other closely related Ostrea species pairs is also uncertain or controversial, including Ostrea edulis and Ostrea angasi, Ostrea conchaphila and Ostrea lurida, and Ostrea permollis and Ostrea puelchana (Coan et al. 2000, Kenchington et al. 2002, Hurwood et al. 2005, Shilts et al. 2007, Polson et al. 2009, Salvi et al. 2014, Raith et al. 2015, Guo et al. 2018).

More than 30 oyster species have been reported to occur along the coast of China, most of which belong to Crassostrea and Saccostrea, with only two species of Ostrea, Ostrea denselamellosa and Ostrea circumpicta (Xu & Huang 1993, Li & Qi 1994, Wang et al. 2004, Reece et al. 2008, Xu & Zhang 2008, Wang et al. 2008, 2010, 2013, Hamaguchi et al. 2014, Guo et al. 2018). The large, flat, and round shells with dense plates along the edge, can be easily identified as Ostrea denselamellosa. The occurrence of O. circumpicta in China has not been genetically confirmed.

To identify possible Ostrea species and clarify their taxonomic status, small flat oysters were collected from China, Japan, and the United States to identify possible Ostrea species by genus-specific PCR markers (Wang & Guo 2008) and sequenced for mitochondrial cytochrome oxidase subunit I (COI) and 16S rRNA genes. Phylogenetic and morphology analysis revealed that the Ostrea stentina complex consisted of three independent species: Ostrea equestris from eastern Atlantic, western and eastern Pacific; O. stentina from eastern Atlantic; and a new species Ostrea neostentina sp. nov. from Mediterranean to Asia. This study provides the first record of O. equestris and O. neostentina sp. nov. in China and adds to the understanding of the diversity, distribution, and evolution of Ostrea species.

MATERIALS AND METHODS

Samples and Identification by Multiplex Genus-Specific PCR

Oysters analyzed in this study were small flat oysters collected from five different locations along the southern coast of China, one location in Japan, and one in North Carolina (Fig. 1, Table 1). Oysters from China and Japan were collected and fixed in 95% ethanol and transported to Shellfish Research Laboratory, Institute of Oceanology, Chinese Academy of Sciences. Oysters from North Carolina were from the collection of Haskin Shellfish Research Laboratory, Rutgers University. Samples of Ostrea edulis, Ostrea angasi, and Ostrea lurida were sequenced at Haskin Shellfish Research Laboratory.

Multiplex genus-specific PCR of Wang and Guo (2008) was used to separate Ostrea species from Crassostrea, Saccostrea, and Hyotissa species. The Saccostrea/Ostrea--specific primer 28Soa166r was used to identify Ostrea and Saccostrea species, and the latter was easily identified by shell morphology.

Sequencing

DNA was extracted from the adductor muscle using the TIANamp Genomic DNA Kit (Tiangen Biology, Beijing, China). The COI fragment was amplified with universal primers: LCO 1490, 5'--GGTCAACAAATCATAAAGATATTGG-3' and HCO2198, 5'--TA[DELTA][DELTA]CTTCAGGGTGACCAAAAAATCA-3' (Folmer et al. 1994). The 16S rRNA fragment was amplified with 16sar, 5'--CGCCTGTTTATCAAAAACAT-3' and 16sbr, 5'--CCGGTCTG[DELTA][DELTA]CTCAGATCACGT-3' (Palumbi et al. 1991).

PCR was performed in 25 [micro]L of reaction mixture containing 2 gL 10x PCR buffer, 1.5 mM [Mg.sup.2+], 200 tM dNTPs, 1 U Taq DNA polymerase, and 0.4 [micro]M forward and reverse primers, using the following parameters: an initial denaturation at 94[degrees]C for 5 min, 30 cycles of 94[degrees]C for 30 sec, 48[degrees]C (16S) or 51[degrees]C (COI) for 1 min, and 72[degrees]C for 1 min, with a final extension at 72[degrees]C for 10 min.

PCR products were verified on 1.5% agarose gels containing 0.2 [micro]g/mL ethidium bromide, visualized on a UV transilluminator, and purified using DP214 Universal DNA Purification Kit (Tiangen Biotech). Sequencing was performed in both directions on an ABI 3730 Genetic Analyzer (Applied Biosystems). Sequences obtained in this study were submitted to NCBI (http://www.ncbi.nlm.nih.gov/) under gene accession numbers KY986305-KY986321, MK370354-392 (16S) and KY986322-KY986335, MK370319-353 (COI) (Table 1).

Phylogenetic Analysis

Reported sequences of Ostrea stentina complex species were downloaded from NCBI GenBank, and their location and accession numbers are provided in Table 2. Sequences (COI and 16S rRNA) from the following reference species were also included in the phylogenetic analysis: Ostreola conchaphila (KT317494.1 and FJ768528), Ostrea lurida (KT317504.1 and FJ768559), Ostrea angelica (KT317449.1 and KT317140), Ostrea permollis (DQ226526.1 and AY376606), Ostrea puelchana (DQ226521.1 and AF052073), Ostrea edulis (KJ818235.1 and AF540595), Ostrea denselamellosa (KP067907.1 and HQ660995), Crassostrea gigas gigas (JF700177.1 and JF808180), Crassostrea gigas angulata (KC170323.I and KC170322), and Hyotissa imbricata (AB076917.1 and KC847136). Haplotypes for 16S rRNA and COI sequences were identified using DnaSP 5 software (Librado & Rozas 2009). Sequences used for phylogenetic analysis were aligned using CLUSTAL x (Thompson et al. 1994) and trimmed to the same length with manual adjustments.

Neighbor-joining (NJ) trees were constructed with MEGA 7 (Kumar et al. 2016) using Kimura's two-parameter (K2P) distances. Bayesian analysis was carried out using the program MrBayes V.3.2.6 (Ronquist et al. 2012). The best fitting models GTR + I + G and HKY + G were selected using Modeltest v3.7 (Posada & Crandall 1998) for 16S rRNA and COI, respectively, based on the Akaike information criteria before Bayesian inference (BI) analyses. Analysis for 20 million generations was run, and parameters and trees were sampled every 10,000 generations, discarding 25% of the total sampled trees from the beginning of the chain as burn-in. Bayesian posterior probabilities were obtained from a tree with clade credibility values. The Hyotissa imbricata species was chosen as the outgroup.

Genetic distances among the Ostrea stentina complex and other reference species were calculated using MEGA 7 with the K2P model.

RESULTS

Shell Morphology

Shells of Ostrea stentina complex species collected in this study are presented in Figure 2. All oysters collected, except one specimen from Hong Kong (Al and A2), closely resemble Ostrea aupouria (=virescens) and Ostrea equestris in shell morphology as previously reported (Fig. 3). These oysters are generally small and oval or round in shape, with height greater than length in most specimens. The left valves, which have raised margins and shallow umbonal cavities, adhere to the substrate over a large area. The right valves are flatter than the left valves and have narrow, flat commissural shelves. There are a series of fine crenellates on both sides of the ligament along the edges of the right valves. Interior surfaces of the valves vary from dull-gray to green, depending on the environment. The adductor muscle scar is pointed and usually reniform and with greenish color around it. The umbo of the specimen collected from Hong Kong marked as Al and A2 is closer to posterior, and the adductor muscle scar is rounder than that in other specimens; the adductor muscle scar of A 1 and A2 is similar to that of specimens from the Gulf of Hammamet (Dridi et al. 2008).

Genus Assignment

Multiplex genus-specific primers provided genus assignment of all oysters collected in this study. Amplified products showed that the collected oysters were either Ostrea, Saccostrea, or Dendostrea species (Fig. 4). Shell morphology of Saccostrea and Dendostrea species is distinctive: Saccostrea species have a deep umbonal cavity and internal chomata on both shells, and Dendostrea species have radiating ribs on the surface of the right valve. Thus, Ostrea species were identified with molecular markers and separated from Saccostrea and Dendostrea species based on shell morphology.

Phylogenetic Analyses

From 57 specimens of Ostrea species collected and identified in this study, 49 COI sequences and 57 16S sequences were obtained. After alignment and trimming, 429 bp of 16S and 603 bp of COI sequences were used for phylogenetic analysis. Among the 49 COI sequences, 25 haplotypes were identified (Table 3), and 13 haplotypes were identified among the 57 16S sequences (Table 4). Oysters from China did not share any haplotypes with oysters from North Carolina in either genes.

The NJ trees constructed based on COI and 16S are presented in Figures 5 and 6, respectively, which show the same topology. Haplotypes obtained in this study clustered together with sequences from the Ostrea stentina complex species, Ostrea aupouria, Ostrea equestris, and O. stentina. Within the large cluster, the sequences obtained in this study were closer to O. aupouria and O. equestris than to O. stentina.

According to the phylogenetic trees, the Ostrea stentina complex could be divided into four groups: group 1 includes O. aupouria/stentina sequences from China, Japan, and New Zealand; group 2 includes Ostrea equestris sequences from Gulf of California, Argentina, Florida, and North Carolina; group 3 includes O. stentina sequences from Mar Menor Lagoon in southeastern Spain, eastern Tunisia (Kneiss Islands-Gannouche, Gulf of Hammamet), one from Japan, and one from Hong Kong, China; and group 4 includes O. stentina sequences from Aviles in northern Spain; Portugal; Dakhla Bay, Morocco; and northern Tunisia (Bizerte Lagoon). Except for one specimen from Japan and one from Hong Kong, the groupings on the phylogenetic trees are mostly in accordance with geographical distribution in the Pacific, the Americas, and eastern Atlantic.

Genetic Distances

The K2P genetic distances between the obtained 16S haplotypes (except Hap 13) and Ostrea aupouria/Ostrea equestris were 0.2%-1.0%, smaller than that between the haplotypes and Ostrea stentina (1.0%-1.9%) (Table 5). The largest 16S distance within the O. stentina complex (1.9%) was larger than that between two subspecies Crassostrea gigas gigas and Crassostrea gigas angulata (1.05%-1.32%) (Wang et al. 2010), as well as that among Ostrea lurida, Ostreola conchaphila, and Ostrea angelica (0.5%-1.2%); between Ostrea permollis and Ostrea puelchana (0.2%); and between Ostrea edulis and Ostrea angasi (0.5%) (Table 5).

Cytochrome oxidase subunit I sequences are more variable and provided higher resolution than 16S sequences. The K2P genetic distances between the COI haplotypes collected in this study (except Hap 2) and Ostrea aupouria/Ostrea equestris were 0%-1.2%, smaller than that between the haplotypes and Ostrea stentina (3.3%-4.3%) (Table 6). The largest distance within the O. stentina complex (4.3%) was larger than that between two subspecies Crassostrea gigas gigas and Crassostrea gigas angulata (2.22%-3.37%) (Wang et al. 2010), as well as that between Ostrea permollis and Ostrea puelchana (2.5%), and between Ostrea edulis and Ostrea angasi (1.9%) (Table 7).

Net average genetic distances (Tamura & Nei 1993) among the four groups are presented in Table 7. Giving the substitution rate of 2% (Marko et al. 2010) to 2.4% (Lazoski et al. 2011) for COI, the genetic distances correspond to divergences at about 0.25-0.31 million years ago (Mya) for groups 1 and 2, 0.72-1.04 Mya for groups 1 + 2 and groups 3 + 4, and 0.86-1.03 Mya between groups 3 and 4.

DISCUSSION

Taxonomic Status

Phylogenetic analysis in this study shows that all sequences obtained in this study are closely related and belong to the Ostrea stentina species complex that includes O. stentina, Ostrea equestris, and Ostrea aupouria. The taxonomic status of flat oysters O. stentina, O. equestris, and O. aupouria has been controversial. Kirkendale et al. (2004) examined the phylogenetic relationships between American O. equestris and New Zealand O. aupouria and showed they are synonymous species. Lapegue et al. (2006) recognized the close relationship among O. stentina, O. aupouria, and O. equestris and pointed out that their phylogenetic status deserved attention because of their geographic disjunction in New Zealand, Mexico, Gulf/Atlantic, and Mediterranean/African Atlantic. Shilts et al. (2007) and Salvi et al. (2014) suggested that O. stentina/O. equestris/O. aupouria may belong to a single taxon. Hamaguchi et al. (2017) identified O. stentina in Japan and recognized significant divergence within the O. stentina complex. Guo et al. (2018) suggested that the O. stentina species complex may consist of three species: O. equestris, O. stentina A, and O. stentina B.

Phylogenetic analysis of all available sequences of the Ostrea stentina complex revealed four closely related groups, which supports recent and ongoing speciation of Ostrea oysters (Guo et al. 2018). The genetic distances between groups 1 and 2 are 1.2% in COI and 0.7% in 16S, well below the level expected for interspecific divergence, considering the distances between subspecies Crassostrea gigas gigas and Crassostrea gigas angulata are 2.22%-3.37% in COI and 1.05%-1.32% in 16S (Wang et al. 2010). It has been suggested that oysters with a divergence in COI below 2% should be generally considered as the same species (Guo et al. 2018). Thus, oysters in groups 1 and 2 are accepted as one species. As the name Ostrea equestris (Say, 1834) predated Ostrea aupouria (Dinamani & Beu 1981), oysters of groups 1 and 2 should take the name of O. equestris. All Ostrea oysters collected from China belong to group 1--O. equestris, except for one sequence belonging to group 3, both are the first records for China. A sequence from a specimen from Taiwan submitted to GenBank under the name Ostrea sp. is also O. equestris. Results of this study indicate that O. equestris is widely distributed along the southern coast of China (Fig. 1).

These results greatly expand the known geographic distribution of Ostrea equestris, now including Asian specimens from China, Japan, and New Zealand, as well as American specimens from Argentina, Columbia, Florida, North Carolina, and Gulf of California on the Pacific side. There are clear and significant divergences between the Asian Pacific and American populations. All specimens from the Americas belonged to group 2, and all Asian Pacific specimens belonged to group 1, except for one specimen (16S Hap 11) from Hong Kong, which is clustered with the American samples. Hong Kong is an international hub of ship traffic, and whether the lone specimen represents a foreign introduction requires further studies. The wide distribution of O. equestris in both Atlantic and Pacific oceans, from Argentina, Columbia, the United States, Gulf of California, Japan, China, to New Zealand, is unusual and highlights wide connectivity of Ostrea species across oceans. The species of group 3 also had a wide distribution, ranging from the Mediterranean Sea to Hong Kong and Japan. Such a wide geographic distribution of a single species is rare for Crassostrea species (Guo et al. 2018). The unique biology and life history of these Ostrea species, such as hermaphroditism and small size, may increase the odds of colonization in nonnative environments during range expansion. The equatorial currents and Pacific Islands may be important in the range expansion of O. equestris. The circumglobal distribution of Neopycnodonte cochlear may be related to its deep-sea environment (Harry 1985, Guo et al. 2018). Understanding the wide geographic distribution of oyster species is an important part of marine phylogeography.

Genetic distances in COI among groups 1 + 2, group 3, and group 4 ranged from 3.5% to 4.2%, well above the 2% level commonly accepted for interspecific divergence (Guo et al. 2018) and the divergence between subspecies Crassostrea gigas gigas and Crassostrea gigas angulata (2.22%-3.37%) (Wang et al. 2010) and between Ostrea permollis and Ostrea puelchana (2.5%). Based on their significant divergence and their mostly distinctive distributions, groups 1 + 2, 3, and 4 should be accepted as separate species. As groups 1 and 2 are accepted as Ostrea equestris, groups 3 and 4 are considered as two species independent of O. equestris. This finding is consistent with the previous suggestion of three species in the Ostrea stentina species complex: O. equestris, O. stentina A, and O. stentina B (Guo et al. 2018).

The original record of Ostrea stentina was from Mediterranean Corsica described by Payraudeau (1826). It is characterized by strongly crenellated valves with the teeth fitting into each other. Shells of O. stentina holotype is shown in Figure 3M, and shells reported by Huber (2010), L1 and L2 in Figure 3, somewhat resemble the original description. In both reports, shells of O. stentina are characterized by regularly waved edges. Shells of group 3 oysters collected from the Gulf of Hammamet (01-04 in Figure 3; Dridi et al. 2008) and China (Fig. 2A1, A2) are clearly different from that of O. stentina holotype and Huber's specimen and should be accepted as a new species. Thus, group 3 is accepted as a new species and named Ostrea neostentina sp. nov. for its close relationship with O. stentina.

Systematics and Description of the New Species Ostrea neostentina sp. nov.

Order: Ostreida (Ferussac, 1822).

Family: Ostreidae (Rafinesque, 1815).

Subfamily: Ostreinae (Rafinesque, 1815).

Genus: Ostrea (Linnaeus, 1758).

Ostrea neostentina sp. nov. new species (Fig. 2A1, A2 and 301-04).

Type Locality

Hong Kong, China.

Material Examined

Holotype, 1 shell (SL 16 x SW 7 x SH 27 mm) (deposited in the Marine Biological Museum of the Chinese Academy of Sciences, accession number: MBM286577).

Description

Shells are elongate-ovate and small, with height greater than length. Exterior color is mainly pewter or close to the color of stone. The left valves adhere to the substrate over a large area. The interior of the right valve is green, and in the left valve, it is white with green. There are a series of fine crenellates on both sides of the ligament along the edges of the right valves. The adductor muscle scar is rounder and larger than that in other species. The umbo is closer to posterior.

Etymology

The species is closely related to Ostrea stentina, but a new species.

Habitat

The holotype specimen was collected by trawling from shallow water within 20 m depth.

Distribution

It is found in Hong Kong of China, Kagoshima and Ibusuki in Japan, and Gulf of Hammamet in Tunisia.

It should be noted that shells of group 4 (Fig. 3N1-N3, reference), which is widely accepted as Ostrea stentina, are quite different from the shells of the holotype (Fig. 3M). Therefore, the currently recognized O. stentina, as represented by multiple sequences in GenBank, may not be the original O. stentina of Payraudeau (1826), although they are from the same area. It is possible that there is another small flat oyster that matches the O. stentina holotype in shell morphology. If so, the currently accepted name O. stentina should be reconsidered.

Evolutionary History

According to the Paleobiology Database at Fossil Works (http://fossilworks.org/), Ostrea stentina was found in Algeria and Morocco during Miocene and in Angola, Italy, and Namibia during Quaternary. In western Atlantic, Ostrea equestris was in Panama and Venezuela during Miocene and in Brazil, Jamaica, and the United States (Florida) during Quaternary. The divergence time among the groups estimated based on COI sequence dated back to Pleistocene: 0.25-0.31 Mya between groups 1 and 2, 0.72-1.04 Mya between groups 1/2 and 3/4, and 0.86-1.03 Mya between groups 3 and 4. It is consistent with the suggestion of recent and ongoing speciation of living oysters (Guo et al. 2018). The eastern Atlantic and west Mediterranean region where groups 3 and 4 diverged first might be the ancestral home of the O. stentina complex. Over time, they expanded to east Mediterranean, western Atlantic, and Pacific via ocean currents, and subsequent isolation led to the speciation of Ostrea neostentina sp. nov. and O. equestris. Dridi et al. (2008) held that the Siculo-Tunisian Strait between eastern and northern Tunisia might act as a barrier effecting the differentiation of O. stentina and the speciation of O. neostentina sp. nov. Ostrea neostentina sp. nov. occurs in both eastern and western Mediterranean and also in Hong Kong and Japan. Its occurrence in Asia may be due to its range expansion or human activities as its haplotype diversity in Hong Kong and Japan is low. It is likely that other populations of O. neostentina exist along the coast of the Indian Ocean.

In conclusion, this study identified small Ostrea species from China, Japan, and the United States and clarified the taxonomy of the Ostrea stentina species complex. Phylogenetic analysis confirms that the O. stentina species complex consisted of three species: Ostrea equestris from the Americas and Asia, O. stentina from eastern Atlantic, and a new species Ostrea neostentina sp. nov. from the Mediterranean Sea and Asia. This study adds to the understanding of the diversity, global distribution, and evolution of Ostrea species, providing support for recent and ongoing speciation of oysters (Guo et al. 2018). It also highlights the need for global surveys to fully understand the diversity and evolution of living oysters.

ACKNOWLEDGMENTS

We thank Ruffian Liu, Tao Zhang, Peizhen Ma, Yongqiang Li, and Dong Dong for helping with sampling. This work was partly supported by grants of National Science Foundation of China (No. 41776179), Strategic Priority Research Program of the Chinese Academy of Sciences (XDA 23050403, 23050304), the Earmarked Fund for Modem Agro-industry Technology Research System (CARS-47), Project of the S & T Basic Work (2014FY110500), Taishan Overseas Scholar Program (1004475), US NOAA CBO Non-native Oyster Research Program (NA04NMF4570424), and Rutgers University (NJ32920).

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LISHA HU, (1,2,3) HAIYAN WANG, (1,2,4*) ZHEN ZHANG, (1,2) CUI LI (1,2) AND XIMING GUO (4*)

(1) Department of Marine Organism Taxonomy and Phylogeny, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao, Shandong 266071, China; (2) Center for Ocean Mega-Science, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, China; (3) University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, China; (4) Haskin Shellfish Research Laboratory, Department of Marine and Coastal Sciences, Rutgers University, 6959 Miller Avenue, Port Norris, NJ 08349

(*) Corresponding authors. E-mails: haiyanwang@qdio.ac.cn or xguo@hsrl.rutgers.edu

DOI: 10.2983/035.038.0210
TABLE 1.
Location, sample size, and sequence accession numbers for oysters
collected in this study.

Abbreviation   Sampling site              GPS coordinates

1. FM         Full Moon Island,  135.336[degrees] E, 33.69[degrees] N
                Japan
2. ST         Shantou, China     116.84[degrees] E, 23.34[degrees] N
3. DY         Daya Bay, China    114.92[degrees] E, 23[degrees] N
4. XG         Hong Kong, China   113.968[degrees] E, 22.32[degrees] N
5. BH         Bohe, China        111.03[degrees] E, 21.3[degrees] N
6. FJ         Fengjiacun, China  110.74[degrees] E, 19.41[degrees] N
7. NC         North Carolina      75.64[degrees] W, 35.69[degrees] N

Abbreviation  Number of  GenBank numbers of submitted sequences
              specimens         COI               16S

1. FM             2       1 (KY986322)       2 (KY986305,09)
2. ST             1       1 (KY986328)       1 (KY986306)
3. DY            25      24 (KY986326;      25 (KY986307,10;
                         MK370331-353)      MK370370-392)
4. XG            16      12 (MK370319-330)  16 (MK370354-369)
5. BH             3       2 (KY986323,27)    3 (KY986308,19-20)
6. FJ             3       2 (KY986324-25)    3 (KY986311-12)
7. NC             7       7 (KY986329-35)    7 (KY986313-18,21)

TABLE 2.
Location and sequence accession numbers of Ostrea stentina complex
species from GenBank.

Species           Abbreviation          Sites

Ostrea stentina   8. WK         Wakayama, Kemi, Japan
                  9. KI         Kagoshima, Ibusuki, Japan
Ostrea sp.        10. TW        Taiwan, China
Ostrea aupouria   11. HG        Hauraki Gulf, New Zealand
Ostrea equestris  12. BM        Bahia Magdalena, Mexico
                  13. LC        Los Cabos, Mexico
                  14. SR        Skidaway River, GA
                  15. CK        Cedar Key, FL
                  16. SB        Sprigger Bank, FL
                  17. BP        Big Pine Key, FL
                  18. WS        West Summerland Key, FL
                  19. FK        Fiesta Key, FL
                  20. NB        Neguange Bay, Colombia
                  21. SM        San Matias Gulf, Argentina
O. stentina       22. AS        Aviles, Spain
                  23. MM        Mar Menor Lagoon, Spain
                  24. KN        Kneiss Islands, Tunisia
                  25. GH        Gannouche, Tunisia
                  26. SA        Sado estuary, Portugal
                  27. MI        Mira estuary, Portugal
                  28. MO        Dakhla Bay, Morocco
                  29. HM        Gulf of Hammamet, Tunisia
                  30. BI        Bizerte Lagoon, Tunisia

Species           Number of sequences (GenBank
                         accession number)
                        COI              16S

Ostrea stentina   3 (LC051582-84)   3 (LC051572-74)
                  7 (LC051585-91)   7 (LC051575-81)
Ostrea sp.        2 (JQ027291-92)   -
Ostrea aupouria   8 (AF112288,      1 (AF052064)
                  DQ226533,
                  AY376627-32)
Ostrea equestris  1 (KT317503)      1 (KT317198)
                  7 (KT317496-502)  7 (KT317191-97)
                  6 (AY376614-19)   -
                  7 (AY376620-26)   -
                  1 (AY376613)      -
                  1 (DQ226522)      1 (AF052074)
                  5 (AY376607-11)   2 (AY376603-04)
                  1 (AY376612)      -
                  1 (DQ226523)      -
                  5 (DQ640078-82)   1 (DQ640402)
O. stentina       3 (KJ818237-39)   1 (KJ818210)
                  3 (DQ226515-17)   1 (DQ180744)
                  1 (DQ313181)      1 (DQ313178)
                  1 (DQ313181)      1 (DQ313178)
                  1 (DQ313182)      1 (DQ313179)
                  1 (DQ313182)      1 (DQ313179)
                  1 (DQ313183)      1 (DQ313180)
                  -                 7 (EU409052-57,63)
                  -                 5 (EU409058-62)

DQ313182 is too short for phylogenic analysis and not used in the
analysis.

TABLE 3.
Distribution of COI haplotypes among sampling sites.

Haplotype  FM  ST  DY  XG  BH  FJ  NC
   1       1   0   7   5   1   0   0
   2       0   0   0   1   0   0   0
   3       0   0   0   1   0   0   0
   4       0   0   3   1   0   0   0
   5       0   0   0   1   0   0   0
   6       0   0   0   1   0   0   0
   7       0   0   0   1   0   0   0
   8       0   0   4   1   1   0   0
   9       0   0   2   0   0   0   0
  10       0   0   1   0   0   0   0
  11       0   0   2   0   0   0   0
  12       0   0   1   0   0   0   0
  13       0   0   1   0   0   0   0
  14       0   0   1   0   0   0   0
  15       0   0   2   0   0   1   0
  16       0   0   1   0   0   0   0
  17       0   1   0   0   0   0   0
  18       0   0   0   0   0   1   0
  19       0   0   1   0   0   0   0
  20       0   0   0   0   0   0   1
  21       0   0   0   0   0   0   1
  22       0   0   0   0   0   0   2
  23       0   0   0   0   0   0   1
  24       0   0   0   0   0   0   1
  25       0   0   0   0   0   0   1

TABLE 4.
Distribution of 16S haplotypes among sampling sites.

Hap  FM  ST  DY  XG  BH  FJ  NC

 1   0   0    0  0   0   0   5
 2   0   0    0  0   0   0   1
 3   0   0    0  0   0   0   1
 4   1   1    6  4   1   0   0
 5   0   0    0  0   1   0   0
 6   0   0    2  1   1   0   0
 7   0   0    2  1   0   1   0
 8   1   0   12  5   0   2   0
 9   0   0    2  2   0   0   0
10   0   0    1  0   0   0   0
11   0   0    0  1   0   0   0
12   0   0    0  1   0   0   0
13   0   0    0  1   0   0   0

TABLE 5.
K2P genetic distances in partial 16S rDNA sequence among oyster species
studied.

                                           1      2      3      4

1. Hap 2*
2. Hap 4*                                0.010
3. Hap 8*                                0.007  0.002
4. Hap 12*                               0.010  0.005  0.002
5. Hap 13*                               0.012  0.010  0.012  0.015
6. Ostrea aupouria AF052064              0.010  0.005  0.002  0.005
7. Ostrea equestris AY376603             0.002  0.007  0.005  0.007
8. Ostrea stentina EU409056              0.012  0.012  0.015  0.017
9. O. stentina KJ818210                  0.014  0.012  0.010  0.012
10. Ostrea lurida 2*                     0.035  0.035  0.032  0.035
11. Ostrea conchaphila KT317144          0.032  0.029  0.032  0.034
12. Ostrea angelica KT317133             0.022  0.024  0.022  0.024
13. Ostrea permollis AF052075            0.045  0.047  0.045  0.047
14. Ostrea puelchana AF052073            0.040  0.042  0.040  0.042
15. Ostrea edulis 3*                     0.080  0.080  0.080  0.077
16. Ostrea angasi*                       0.086  0.086  0.086  0.083
17. Crassostrea gigas gigas JF808180     0.192  0.192  0.192  0.189
18. Crassostrea gigas angulata KC170322  0.185  0.185  0.185  0.182
19. Hyotissa imbricata KC847136          0.340  0.339  0.343  0.347

                                           5      6      7      8

1. Hap 2*
2. Hap 4*
3. Hap 8*
4. Hap 12*
5. Hap 13*
6. Ostrea aupouria AF052064              0.015
7. Ostrea equestris AY376603             0.012  0.007
8. Ostrea stentina EU409056              0.002  0.017  0.012
9. O. stentina KJ818210                  0.017  0.012  0.015  0.019
10. Ostrea lurida 2*                     0.042  0.035  0.032  0.042
11. Ostrea conchaphila KT317144          0.034  0.034  0.034  0.034
12. Ostrea angelica KT317133             0.027  0.024  0.022  0.027
13. Ostrea permollis AF052075            0.050  0.047  0.045  0.050
14. Ostrea puelchana AF052073            0.045  0.042  0.040  0.045
15. Ostrea edulis 3*                     0.083  0.083  0.080  0.083
16. Ostrea angasi*                       0.088  0.089  0.086  0.088
17. Crassostrea gigas gigas JF808180     0.189  0.195  0.189  0.189
18. Crassostrea gigas angulata KC170322  0.179  0.189  0.182  0.179
19. Hyotissa imbricata KC847136          0.335  0.347  0.343  0.335

                                           9     10     11      12

1. Hap 2*
2. Hap 4*
3. Hap 8*
4. Hap 12*
5. Hap 13*
6. Ostrea aupouria AF052064
7. Ostrea equestris AY376603
8. Ostrea stentina EU409056
9. O. stentina KJ818210
10. Ostrea lurida 2*                     0.040
11. Ostrea conchaphila KT317144          0.039  0.012
12. Ostrea angelica KT317133             0.027  0.005  0.010
13. Ostrea permollis AF052075            0.042  0.035  0.037  0.029
14. Ostrea puelchana AF052073            0.037  0.030  0.032  0.024
15. Ostrea edulis 3*                     0.083  0.069  0.069  0.061
16. Ostrea angasi*                       0.088  0.075  0.074  0.067
17. Crassostrea gigas gigas JF808180     0.205  0.186  0.189  0.189
18. Crassostrea gigas angulata KC170322  0.199  0.179  0.182  0.183
19. Hyotissa imbricata KC847136          0.350  0.331  0.314  0.331

                                          13     14     15     16

1. Hap 2*
2. Hap 4*
3. Hap 8*
4. Hap 12*
5. Hap 13*
6. Ostrea aupouria AF052064
7. Ostrea equestris AY376603
8. Ostrea stentina EU409056
9. O. stentina KJ818210
10. Ostrea lurida 2*
11. Ostrea conchaphila KT317144
12. Ostrea angelica KT317133
13. Ostrea permollis AF052075
14. Ostrea puelchana AF052073            0.002
15. Ostrea edulis 3*                     0.069  0.064
16. Ostrea angasi*                       0.074  0.069  0.005
17. Crassostrea gigas gigas JF808180     0.202  0.200  0.186  0.183
18. Crassostrea gigas angulata KC170322  0.195  0.193  0.186  0.183
19. Hyotissa imbricata KC847136          0.352  0.353  0.341  0.350

                                          17    18

1. Hap 2*
2. Hap 4*
3. Hap 8*
4. Hap 12*
5. Hap 13*
6. Ostrea aupouria AF052064
7. Ostrea equestris AY376603
8. Ostrea stentina EU409056
9. O. stentina KJ818210
10. Ostrea lurida 2*
11. Ostrea conchaphila KT317144
12. Ostrea angelica KT317133
13. Ostrea permollis AF052075
14. Ostrea puelchana AF052073
15. Ostrea edulis 3*
16. Ostrea angasi*
17. Crassostrea gigas gigas JF808180
18. Crassostrea gigas angulata KC170322  0.005
19. Hyotissa imbricata KC847136          0.403  0.399

Bold numbers refer interspecific distances among Ostrea stentina
complex species.
(*) Sequences obtained in this study. Thirteen haplotypes were
identified in this study, and five are presented here. Hap 13 is Ostrea
neostentina and Hap 2/4/8/12 are Ostrea equestris.

TABLE 6.
K2P genetic distances in partial COI sequence among oyster species
studied.

                                           1      2      3      4

1. Hap 1*
2. Hap 2*                                0.036
3. Hap 3*                                0.008  0.038
4. Hap 4*                                0.003  0.040  0.012
5. Hap 5*                                0.002  0.038  0.010  0.005
6. Ostrea sp. JQ027291                   0.000  0.036  0.008  0.003
7. Ostrea aupouria DQ640080              0.008  0.034  0.010  0.012
8. Ostrea stentina DQ226517              0.034  0.002  0.036  0.038
9. O. stentina KJ818237                  0.038  0.040  0.043  0.041
10. Ostrea lurida KT317504               0.135  0.133  0.137  0.135
11. Ostrea conchaphila KT317494          0.135  0.133  0.137  0.135
12. Ostrea angelica KT317449             0.135  0.146  0.137  0.130
13. Ostrea permollis DQ226526            0.116  0.121  0.114  0.112
14. Ostrea puelchana DQ226521            0.120  0.125  0.118  0.116
15. Ostrea edulis KJ818235               0.212  0.223  0.209  0.209
16. Ostrea angasi AF540598               0.222  0.233  0.214  0.220
17. Crassostrea gigas gigas JF700177     0.282  0.282  0.282  0.282
18. Crassostrea gigas angulata KC170323  0.285  0.280  0.285  0.285
19. Hyotissa imbricata AB076917          0.532  0.509  0.538  0.532

                                           5      6      7     8

1. Hap 1*
2. Hap 2*
3. Hap 3*
4. Hap 4*
5. Hap 5*
6. Ostrea sp. JQ027291                   0.002
7. Ostrea aupouria DQ640080              0.010  0.008
8. Ostrea stentina DQ226517              0.036  0.034  0.033
9. O. stentina KJ818237                  0.040  0.038  0.040  0.042
10. Ostrea lurida KT317504               0.137  0.135  0.133  0.131
11. Ostrea conchaphila KT317494          0.137  0.135  0.129  0.131
12. Ostrea angelica KT317449             0.137  0.135  0.133  0.144
13. Ostrea permollis DQ226526            0.118  0.116  0.114  0.119
14. Ostrea puelchana DQ226521            0.122  0.120  0.118  0.123
15. Ostrea edulis KJ818235               0.214  0.212  0.215  0.220
16. Ostrea angasi AF540598               0.225  0.222  0.220  0.230
17. Crassostrea gigas gigas JF700177     0.279  0.282  0.277  0.279
18. Crassostrea gigas angulata KC170323  0.282  0.285  0.279  0.277
19. Hyotissa imbricata AB076917          0.538  0.532  0.521  0.504

                                           9     10     11     12

1. Hap 1*
2. Hap 2*
3. Hap 3*
4. Hap 4*
5. Hap 5*
6. Ostrea sp. JQ027291
7. Ostrea aupouria DQ640080
8. Ostrea stentina DQ226517
9. O. stentina KJ818237
10. Ostrea lurida KT317504               0.133
11. Ostrea conchaphila KT317494          0.133  0.002
12. Ostrea angelica KT317449             0.124  0.098  0.098
13. Ostrea permollis DQ226526            0.121  0.136  0.140  0.139
14. Ostrea puelchana DQ226521            0.124  0.124  0.129  0.145
15. Ostrea edulis KJ818235               0.210  0.192  0.197  0.190
16. Ostrea angasi AF540598               0.225  0.192  0.197  0.197
17. Crassostrea gigas gigas JF700177     0.279  0.287  0.284  0.274
18. Crassostrea gigas angulata KC170323  0.271  0.288  0.285  0.274
19. Hyotissa imbricata AB076917          0.532  0.515  0.520  0.542

                                          13     14     15     16

1. Hap 1*
2. Hap 2*
3. Hap 3*
4. Hap 4*
5. Hap 5*
6. Ostrea sp. JQ027291
7. Ostrea aupouria DQ640080
8. Ostrea stentina DQ226517
9. O. stentina KJ818237
10. Ostrea lurida KT317504
11. Ostrea conchaphila KT317494
12. Ostrea angelica KT317449
13. Ostrea permollis DQ226526
14. Ostrea puelchana DQ226521            0.025
15. Ostrea edulis KJ818235               0.212  0.194
16. Ostrea angasi AF540598               0.204  0.187  0.019
17. Crassostrea gigas gigas JF700177     0.298  0.289  0.308  0.294
18. Crassostrea gigas angulata KC170323  0.287  0.287  0.314  0.306
19. Hyotissa imbricata AB076917          0.519  0.492  0.542  0.542

                                          17     18

1. Hap 1*
2. Hap 2*
3. Hap 3*
4. Hap 4*
5. Hap 5*
6. Ostrea sp. JQ027291
7. Ostrea aupouria DQ640080
8. Ostrea stentina DQ226517
9. O. stentina KJ818237
10. Ostrea lurida KT317504
11. Ostrea conchaphila KT317494
12. Ostrea angelica KT317449
13. Ostrea permollis DQ226526
14. Ostrea puelchana DQ226521
15. Ostrea edulis KJ818235
16. Ostrea angasi AF540598
17. Crassostrea gigas gigas JF700177
18. Crassostrea gigas angulata KC170323  0.026
19. Hyotissa imbricata AB076917          0.578  0.573

Bold numbers refer interspecific distances among Ostrea stentina
complex species.
(*) Sequences obtained in this study. Twenty-five haplotypes were
identified in this study, and five are presented here. Hap 2 is Ostrea
neostentina and Hap 1/3/4/5 are Ostrea equestris.

TABLE 7.
Net average K2P genetic distances in COI (below diagonal) and 16S
(above diagonal) among Ostrea stentina complex species groups: 1 & 2,
Ostrea equestris; 3, Ostrea neostentina; and 4, Ostrea stentina.

Groups   1      2       3      4

  1     -      0.007  0.015  0.012
  2     0.012  -      0.013  0.015
  3     0.038  0.035  -      0.019
  4     0.041  0.042  0.041  -
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Author:Hu, Lisha; Wang, Haiyan; Zhang, Zhen; Li, Cui; Guo, Ximing
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